Sponsored by Rev. Richard Mather,[3] Dunster emigrated to Boston, Massachusetts in 1640. When Nathaniel Eaton was dismissed in 1639 as master of the recently established Harvard College, in Cambridge, Massachusetts, Dunster was appointed as his successor. Thus on August 27, 1640 Dunster became the first president of Harvard. (For a discussion of Dunster's choice of the title "president" see President#Title.) He modeled Harvard's educational system on that of the English schools such as Eton College and Cambridge University. He set up as well as taught Harvard's entire curriculum alone for many years, graduating the first college class in America, the Class of 1642. From 1649-1650 Dunster also served as interim pastor at The First Parish in Cambridge until the accession of Jonathan Mitchel.[4] Historians have generally treated Dunster well in terms of his theological beliefs and educational abilities. Samuel Eliot Morison, the best-known historian of Harvard's history, wrote that Harvard College "might have followed her first patron to an early death and oblivion but for the faith, courage and intelligence of Henry Dunster."[5] Dunster held Harvard together financially during a difficult economic downturn in New England that began soon after his arrival. He later had some conflict with the college's treasurer, Thomas Danforth, who called him the "de facto treasurer.".[6] However, Dunster indeed was the "de facto treasurer" of Harvard for nearly a decade. With the approval of the General Court of Massachusetts Bay, he later set up the first corporation charter in America, the Charter of 1650, and named Danforth as the new treasurer.[7] The corporate charter that Dunster established governs Harvard University to this day—an astounding testament to his leadership and governing skills. On December 6, 2010, Harvard announced its intention to expand the membership of the Corporation from a body of seven members (as first set up by Dunster) to thirteen members.[8]

When Dunster abandoned the Puritan view of infant baptism in favor of believer's baptism in 1653/54, he provoked a controversy that highlighted two distinct approaches to dealing with dissent in the Massachusetts Bay Colony. The colony's Puritan leaders, whose own religion was born of dissent from mainstream Church of England, generally worked for reconciliation with members who questioned matters of Puritan theology but responded much more harshly to outright rejection of Puritanism. Dunster's conflict with the colony's magistrates began when he failed to have his infant son baptized, believing that only adults should be baptized. Earnest efforts to restore Dunster to Puritan orthodoxy failed, and his heterodoxy proved untenable to colony leaders who had entrusted him, in his job as Harvard's president, to uphold the colony's religious mission. Thus, he represented a threat to the stability of society. Dunster exiled himself in 1654/55 and moved to nearby Plymouth Colony to become the minister of the First Church in Scituate, Massachusetts. Dunster died on February 27, 1659 (or 1658 – Old Style calendar).[9]

Dunster married twice; both his wives were named Elizabeth. His first wife was Elizabeth (Harris) Glover, the widow of Joseph Glover. They married on June 21, 1641. She died in 1643, leaving Dunster with land and property, including the first printing press in the colony, and leaving him shared responsibility for her estate and her five children by her first marriage. Dunster married Elizabeth Atkinson (1627–1690) in 1644. Together they had five children.